Hepatitis C is one of the most widespread infectious diseases in the world. About 170 million people are infected with hepatitis C virus (HCV) worldwide with a yearly incidence of 3-4 million. While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and posttranslationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 6 major HCV genotypes (genotypes 1-6) have been identified, which differ in nucleotide and amino acid sequence composition by 31-35% (Bukh et al. 1993). In addition, there are numerous subtypes (a, b, c, etc.). Genotype 5a is only commonly found in South Africa where it represents 30% of the HCV infections detected. HCV genotype 5a is found in less than 3% of the infected population in countries with sporadic cases. However, recently there have been reports on regional high prevalence of HCV genotype 5a, in the central part of France. Genotyping of patients in this region studied from (1996-2002) showed a prevalence of 14%, making genotype 5a the 3rd most frequent subtype, and interestingly the patients were from a settled, older part of the population where the reason was less likely to be transmission from intravenous drug use or travel to endemic countries. In another recent study of patients in Flandern, Belgium, a prospective study from 2001 to 2004 showed that genotype 5a was the second most prevalent genotype in that area with 30% prevalence compared to 50% prevalence of genotype 1.
The only approved therapy for HCV comprises a combination therapy with interferon and ribavirin. Such therapy is expensive and associated with severe side-effects and contraindications. Sustained viral response can be achieved in only about 55% of treated patients in general, in 85-90% of patients infected with genotypes 2 and 3 and only in 40-50% of patients infected with other genotypes. There is no vaccine against HCV.
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines. Full-length consensus cDNA clones of HCV strain H77 (genotype 1a) and J6 (genotype 2a) shown to be infectious in the chimpanzee model were apparently not infectious in vitro. Replicon systems permitted the study of HCV RNA replication in cell culture using the human liver hepatoma cell line Huh7 but were dependent on adaptive mutations that were deleterious for infectivity in vivo.
In 2001, a genotype 2a isolate (JFH1) was described (Kato et al., 2001), which yielded high RNA titers in the replicon system without adaptive mutations (Kato et al., 2003).
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells (Wakita et al., 2005) (Zhong et al., 2005)
At the same time, Lindenbach et al. demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (C, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic (Lindenbach et al., 2005). Cell culture derived J6/JFH viruses were apparently fully viable in vivo.
Despite the importance of the described cell culture systems they represent only a single subtype (genotype 2a) of HCV. It is important to develop cell culture systems for representative strains of other HCV genotypes, since neutralizing antibodies are not expected to cross-neutralize all genotypes and new specific antiviral compounds might have differential efficiencies against different genotypes. For the genotype specific study of the function of the structural proteins, p7 and NS2 as well as related therapeutics such as neutralizing antibodies, fusion inhibitors, ion-channel blockers and protease inhibitors, it would be sufficient to construct intergenotypic recombinant viruses in analogy to J6/JFH.
Pietschmann et al. 2006 disclose construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus recombinants. The authors created a series of recombinant genomes allowing production of infectious genotype 1a, 1b, 2a and 3a particles by constructing hybrid genomes between the JFH1 isolate and the HCV isolates: H77 (genotype 1a), Con1 (genotype 1b), J6 (genotype 2a) and 452 (genotype 3a) respectively. Thus, disclosing both genotypes completely different from the genotype disclosed in the present application and relating to completely different strains of origin.
The infectious titers of the 1a, 1b and 3a genotypes disclosed in Pietschmann et al. 2006 are not at a level sufficiently high for practical utilization in functional analysis, drug and vaccine development or other applications. For such applications, including screening of potential drugs and development of potential vaccine candidates, the skilled person will know that infectivity titers below 103 TCID50/mL contain insufficient amounts of infectious virus.
Accordingly, the study does not attempt cell culture adaptation of the genotype recombinants, e.g. by serial passage of cell culture derived viruses to naïve cells and it is not investigated whether adaptive mutations develop after transfection in cell culture. In fact, Pietschmann et al does not even provide any sequence data of the virus produced in the cell culture.